Laboratory Evaluation of Sperm Chromatin: TUNEL Assay

Transcription

1 See discussions, stats, and author profiles for this publication at: Laboratory Evaluation of Sperm Chromatin: TUNEL Assay Article January 2014 DOI: / _14 CITATIONS 5 READS 1,361 2 authors: Rakesh K Sharma Cleveland Clinic 458 PUBLICATIONS 10,148 CITATIONS Ashok Agarwal Cleveland Clinic 1,829 PUBLICATIONS 31,891 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: SDF testing View project Research Profile View project All content following this page was uploaded by Ashok Agarwal on 27 January The user has requested enhancement of the downloaded file.

2 Laboratory Evaluation of Sperm 14 Chromatin: TUNEL Assay Rakesh Sharma and Ashok Agarwal Abstract Routine semen analysis is unable to assess alterations in sperm chromatin organization such as DNA damage. Because fertility is based not only on the absolute number of spermatozoa but also on their functional capability, methods for exploring sperm DNA stability and integrity are being used to evaluate fertility disorders. A large number of direct and indirect tests that measure sperm DNA damage have been developed. This chapter focuses on one of those tests the terminal deoxytransferase mediated deoxyuridine triphosphate (dutp) nick end-labeling or TUNEL assay which is increasingly being used in many laboratories. Keywords triphosphate assay One of the possible causes of infertility in men with normal semen parameters is abnormal sperm DNA. Fortunately, a number of sperm function tests are available to assess sperm DNA integrity. One of the most commonly used tests is the R. Sharma ( ) Andrology Laboratory and Center for Reproductive Medicine, Glickman Urological and Kidney Institute, OB-GYN and Women s Health Institute, Cleveland Clinic, Cleveland, OH, USA Sharmar@ccf.org terminal deoxytransferase mediated deoxyuridine triphosphate (dutp) nick end-labeling assay, which is otherwise called TUNEL. This test identifies in situ DNA strand breaks resulting from apoptotic signaling cascades by labeling the 3 -hydroxyl (3 -OH) free ends with a fluorescent label. The fluorescence, which is proportional to the number of strand breaks, can be measured either with microscopy or with flow cytometry. This chapter discusses the TUNEL assay in detail, including clinical protocols, clinical outcomes, and future strategies aimed at optimizing this test and increasing its application as the test of choice in clinical andrology. A. Zini and A. Agarwal (eds.), Sperm Chromatin: Biological and Clinical Applications in Male Infertility and Assisted Reproduction, DOI / _14, Springer Science+Business Media, LLC

3 202 R. Sharma and A. Agarwal Mechanisms of Sperm DNA Damage When sperm DNA is damaged, infertility, miscarriage, and birth defects in offspring can occur [1]. The main cause of sperm DNA damage is oxidative stress [2 5]. Oxidative stress occurs when levels of reactive oxygen species (ROS) increase, when levels of antioxidant decrease, or both. A number of factors can lead to oxidative stress, including infection (viral or bacterial), exposure to xenobiotics, and tobacco and alcohol consumption. DNA fragmentation may also occur during spermiogenesis. During this process, torsional stress increases, as DNA is condensed and packaged into the differentiating sperm head. Endogenous endonucleases (topoisomerases) may induce DNA fragmentation as a way of relieving this stress [6, 7]. Spermatozoa are transcriptionally and translationally inactive and cannot undergo conventional programmed cell death or regulated cell death called apoptosis but are capable of exhibiting some of the hallmarks of apoptosis including caspase activation and phosphatidylserine exposure on the surface of the sperm. This form of apoptosis is termed as abortive apoptosis [8, 9]. Sperm cells are able to repair some DNA damage during spermatogenesis, but once they mature, they lose this innate ability [10, 11]. Therefore, posttesticular sperm are more vulnerable to DNA damage. Studies show that DNA damage is lowest in testicular sperm and that it increases in epididymal and ejaculated sperm [12 15]. Measuring Sperm DNA Damage with TUNEL Sperm DNA damage can be assessed with a number of techniques that measure different aspects of DNA damage (Table 14.1). Each assay has its own advantages and disadvantages (Table 14.2). One of the most commonly used assays is the TUNEL assay. The quantity of DNA 3 -OH free ends can be assessed in spermatozoa using this assay in which the terminal deoxytransferease (TdT) enzyme incorporates a fluorescent UTP at the 3 -OH end, and the fluorescence is proportional to the number of DNA strand breaks (Fig. 14.1). This assay can be run either as a slidebased (fluorescent microscopy) (Fig. 14.2) or flow-cytometry assay [16] (Fig. 14.3, Table 14.3). TUNEL identifies what is termed as real or actual DNA damage that is, damage that has already occurred as opposed to potential damage caused by exposing sperm to denaturing conditions (Table 14.4). All of the assays shown in Table 14.1 have a strong correlation with one another. Unfortunately, none of them are able to selectively differentiate clinically important DNA fragmentation from clinically insignificant fragmentation. The assays also cannot differentiate the DNA nicks that occur normally (physiological) from pathological nicking, nor can they evaluate the genes that may be affected by DNA fragmentation. These assays, including TUNEL, can only determine the amount of DNA fragmentation that occurs with the assumption that higher levels of DNA fragmentation are pathological. Measurement of DNA Damage in Spermatozoa by TUNEL Assay DNA damage can be measured using the TUNEL assay by various protocols such as the following: 1. Biotin-d(UTP)/avidin system. 2. BrdUTP/anti-Br-dUTP-FITC system. 3. Fluorescein isothiocynate labeled (FITC) dutp system (In Situ Cell Detection kit, Catalog No , Roche Diagnostics GmBH, Mannheim, Germany or Roche Diagnostics, Indianapolis, IN). 4. Apoptosis detection kit (Apo-Direct kit; Catalog No ; BD Pharmingen, San Diego, CA). We describe protocol #3 and #4 because they are commonly used tests for measuring sperm DNA damage in sperm. The detection of sperm DNA fragmentation by flow cytometry and epifluorescence microscopy methods will also be described.

4 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 203 Table 14.1 Basics of common sperm DNA integrity assays Direct assays TUNEL Comet In situ nick translation Indirect assays DNA break detection FISH SCD Acridine orange flow cytometric assays (e.g., SCSA, SDFA) Acridine orange test Basis of assay Adds labeled nucleotides to free DNA ends Template independent Labels SS and DS breaks Electrophoresis of single sperm cells DNA fragments form tail Intact DNA stays in head Alkaline Comet Alkaline conditions, denatures all DNA Identifies both DS and SS breaks Neutral Comet Does not denature DNA Identifies DS breaks, maybe some SS breaks Incorporates biotinylated dutp at SS DNA breaks with DNA polymerase I Template-dependent Labels SS breaks, not DS breaks Denatures nicked DNA Whole genome probes bind to SS DNA Individual cells immersed in garose Denatured with acid then lysed Normal sperm produce halo Mild acid treatment denatures DNA with SS or DS breaks Acridine orange binds to DNA DS DNA (nondenatured) fluoresces green SS DNA (denatured) fluoresces red Flow cytometry counts thousands of cells Same as above, hand-counting of green and red cells Measured parameter % Cells with labeled DNA % Sperm with long tails (tail length, % of DNA in tail) % Cells with incorporated dutp (fluorescent cells) Amount of fluorescence proportional to number of DNA breaks % Sperm with small or absent halos DFI the percentage of sperm with a ratio of red to (red + green) fluorescence greater than the main cell population % Cells with red fluorescence DFI DNA fragmentation index; DS double-stranded; FISH fluorescence in situ hybridization; SCD sperm chromatin dispersion test; SCSA sperm chromatin structure assay; SDFA sperm DNA fragmentation assay; SS single-stranded; TUNEL terminal deoxynucleotidyl transferase-mediated dutp nick end-labeling From Zini and Sigman [17], with permission First, the semen specimen is collected: (a) Ideally, the sample should be collected after a minimum of 48 h and not more than 72 h of sexual abstinence. The name of the patient, period of abstinence, date, and time and place of collection should be recorded on the form accompanying each semen analysis. (b) The sample should be collected in private in a room near the laboratory. If not, it should be delivered to the laboratory within 1 h of collection. (c) The sample should be obtained by masturbation and ejaculated into a clean, wide-mouth plastic specimen cup. Lubricants should not be used to facilitate semen collection.

5 204 R. Sharma and A. Agarwal Table 14.2 Advantages and disadvantages of various DNA integrity assays Direct assays Pros Cons TUNEL Can perform on few sperms Expensive equipment not required Simple and fast High sensitivity Indicative of apoptosis Correlated with semen parameters Associated with fertility Available in commercial kits Thresholds not standardized Variable assay protocols Not specific to oxidative damage Special equipment required (flow cytometer) COMET High Sensitivity Simple and inexpensive Correlates with seminal parameters Small number of cells required Can perform on few sperm Alkaline: identifies all breaks Neutral: may identify more clinically relevant breaks Labor intensive Not specific to oxidative damage Subjectiveness in data acquired No evident correlation in fertility Lack of standard protocols Requires imaging software Variable assay protocols Alkaline: may identify clinically unimportant fragmentation May induce breaks at alkaline-labile sites In situ nick translation Simple Unclear thresholds Indirect assays Less sensitive DNA break detection FISH Can perform on few sperm Limited clinical data SCD Easy, can use bright-field microscopy Limited clinical data Acridine orange flow cytometric assays Many cells rapidly examined Most published studies reproducible Expensive equipment required Small variations in lab conditions affect results Calculations involve qualitative decisions Manual acridine orange test Simple Difficulty with indistinct colors, rapid fading, heterogeneous staining 8-OHdg analysis High specificity Quantitative High sensitivity Correlated with sperm function Associated with fertility Large amount of sample required Introduction of artifacts Special equipment required Lack of standard protocols FISH fluorescence in situ hybridization; SCD sperm chromatin dispersion test; TUNEL terminal deoxynucleotidyl transferase-mediated dutp nick end-labeling From Zini and Sigman [17], with permission (d) Coitus interruptus is not acceptable as a means of collection because it is possible that the first portion of the ejaculate, which usually contains the highest concentration of spermatozoa, will be lost. Moreover, cellular and bacteriological contamination of the sample and the acid ph of the vaginal fluid adversely affect sperm quality. (e) The sample should be protected from extreme temperatures (not less than 20 C and not more than 40 C) during transport to the laboratory. (f) Any unusual collection or condition of the specimen should be noted on the report form. Protocol #3: In Situ Death Detection kit (Roche Diagnostics, Indianapolis, IN) Reagents and Equipment (a) Flow cytometry tubes (12 75 mm) (b) Pipettes and pipette tips (1,000, 100, and 50 L)

6 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 205 Fig Schematic of the TUNEL assay Fig Fluorescence microscopic staining with TUNEL and propidium iodide. TUNEL-positive sperm stain green and TUNEL-negative samples stain red Fig Representative histogram showing (a) TUNEL negative and (b) TUNEL positive sample (c) Serological pipettes (2 and 5 ml) (d) Sperm counting chamber (MicroCell; Concep tion Technologies, San Diego, CA) (e) Paraformaldehyde in phosphate-buffered saline, ph 7.4 (f) Ethanol (70%) In Situ Death Detection Kit 1. Blue vial/cap (Enzyme solution): It contains the terminal deoxynucleotidyl transferase (TdT) enzyme solution. It is 10 concentration and contains 5 50 L aliquots. 2. Violet vial (Label solution): It consists of a nucleotide mixture in a reaction buffer of 1 concentration and has L aliquots. 3. Benchtop centrifuge. 4. Flow cytometer. 5. Phase and epifluorescence microscope. Assay Principle The cleavage of genomic DNA during apoptosis leads to both single-strand breaks (nicks) and double-stranded, low-molecular-weight DNA fragments. These DNA strand breaks can be

7 206 R. Sharma and A. Agarwal Table 14.3 TUNEL positivity using flow cytometry or fluorescence microscopy technique Reference Sample size TUNEL positive (%) Flow cytometry Microscopy Muratori et al. [18] ± 8.00 ( ) Muratori et al. [19] Lopes et al. [20] ± 1.5 (0.5 75) (swim up) Barroso et al. [21] ± 7 (low motility sample) Donnelly et al. [22] (15 60); 18 (7 45) (gradient) Gandini et al. [23] (infertile) and ~2.5 (fertile) Oosterhuis et al. [24] ± 15 (1.3 64) Sergerie et al. [25] 97 15% controls Ramos and Wetzels [26] (controls) Zini et al. [27] (infertile) and 10.2 (fertile) Duran et al. [28] ± 3.5 (pregnancy) vs ± 10.8 (no pregnancy) (gradient separation) Sakkas et al. [29] ( ) Shen et al. [30] 60 ~15 Weng et al. [9] (patient) and 7 (control) with high motility vs. 33 (patient) and ~25 (control) in low motility samples Benchaib et al. [31] (abnormal samples) and 6 7 (normal after gradient separation) Carrell et al. [32] 21 ~38.4 (miscarriages) Erenpreisa et al. [33] (range) (methanol:ethanol fixed) Erenpreiss et al. [34] (4 27) (frozen) Lachaud et al. [35] ± 2.2 (0 h; washed); 7.6 ± 1.1 (0 h; gradient); 1.7 ± 0.8 (0 h; swim up) Tesarik et al. [36] ± 3.7 (patient) and 8.7 ± 3.6 (control) Greco et al. [37] ± 5.1 (in ejaculates) vs. 4.8 ± 3.6 (testicular sperm) Sergerie et al. [38] (patients) vs (controls) Sergerie et al. [38] ± 14.3 (patients) and 13.1 ± 7.3 (controls) Sergerie et al. [38] ± (patients) and 13.1 ± (controls) Stahl et al. [39] (2.5 31) (control) Sepaniak et al. [40] (nonsmokers) and 32 (smokers) Chohan et al. [41] ± 1.3 (infertile) and 11.1 ± 0.9 (fertile) De Paula et al. [42] ± 3.6 (patient) and 5.4 ± 2.7 (control) Aoki et al. [43] ± 4.9 (low P1/P2) and 21.6 ± 1.7 (normal P1/ P2) and 28.3 ± 3.1 (high P1/P2) Spermon et al. [44] ( ) (pretreatment) and 25.0 (10 47) (posttreatment) Dominguez et al. [45] ± ± 10.3 Sakamoto et al. [46] ± 13.6 prevaricocele and 27.5 ± 19.4 (postvaricocele) P1 protamine-1; P2 protamine-2

8 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 207 Table 14.4 Why TUNEL methods for DNA damage is the method of choice? Principal 1. Adds labeled nucleotides to free DNA ends What is measured TUNEL SCSA Comet 2. Template independent 3. Labels SS and DS breaks % Cells with labeled DNA Type of assay Direct Objective 1. Mild acid treatment denatures DNA with SS or DS breaks 2. Acridine orange binds to DNA 3. Double-stranded DNA (nondenatured) fluoresces green Single-stranded DNA (denatured) fluoresces red 4. Flow cytometry counts thousands of cells DFI the percentage of sperm with a ratio of red to (red + green) fluorescence greater than the main cell population Indirect Objective 1. Electrophoresis of single sperm cells 2. DNA fragments form tail 3. Intact DNA stays in head Alkaline Comet 1. Alkaline conditions, denatures all DNA 2. Identifies both DS and SS breaks Neutral Comet 1. Does not denature DNA 2. Identifies DS breaks, maybe some SS breaks % sperm with long tails (tail length, % of DNA in tail) Direct Subjective Ease of assay Many labs run this assay Samples have to be shipped to reference Lab Very few labs perform this assay Instrumentation Flow cytometry Flow cytometry Microscopy Nature of assay TUNEL kit available Only in reference or designated labs Manual, no assay kits available Reference alues Ranges from 10 to 30% Robust, >30% DFI indicative of decreased Clinically useful reference values not pregnancies established Type of samples Fresh or frozen Fresh or frozen Fresh Repeatability of Good Good Poor assay Cost Inexpensive Expensive Inexpensive

9 208 R. Sharma and A. Agarwal identified by labeling the free 3 -OH termini with modified nucleotides in an enzymatic reaction (Fig. 14.1). This occurs in two stages: (1) Labeling of DNA strand breaks with TdT, which catalyzes the polymerization of labeled nucleotides to free 3 -OH DNA ends in a template-independent manner (TUNEL reaction) and (2) Fluorescein isothiocynate (FITC)-dUTP is incorporated into nucleotide polymers, and it can be directly detected and quantified by fluorescence microscopy or flow cytometry. This kit is designed to be a precise, fast, and simple nonradioactive technique to detect and quantify the number apoptotic cells. It is specific as it labels DNA strand breaks generated during apoptosis, which enables the test to discriminate between apoptotic and necrotic cells. Sample Preparation (a) Wash the semen aliquot containing spermatozoa by centrifuging at 800 g at room temperature for 5 min with phosphate-buffered saline. (b) After removing the seminal plasma, wash the pellet twice in PBS with 1% bovine serum albumin (BSA). (c) Suspend the pellet in 100 L of PBS/BSA (1%) and fix in 100 L of 4% paraformaldehyde in PBS (ph 7.4) for 1 h at room temperature by vortexing. (d) Resuspend the pellet in 100 L of PBS and permeabilize with 100 L of 0.1% Triton X 100 in 0.1% sodium citrate in PBS for 2 min in ice. Repeat two washes in PBS/BSA (1%). (e) Preparation of the staining solution One pair of tubes (vial 1: Enzyme solution, (50 L) and vial 2: Label solution (550 L)) is sufficient for staining ten samples. The TUNEL reaction mixture is prepared by adding 50 L of enzyme solution to 450 L of label solution to give a total volume of 500 L. (f) Preparation of negative and positive controls Negative control: Incubate fixed and permeabilized cells with 50 L of label solution (without TdT). Positive control: Incubate fixed and permeabilized cells with DNase I (3 3,000 U/mL in 50 mm Tris-HCl, ph 7.5, 1 mg/ml BSA) for 10 min at 25 C to induce DNA damage. (g) Resuspend the pellet in 50 L of the staining solution for 1 h at 37 C in the dark and mix them. (h) After staining, rinse twice in PBS/BSA (1%) and resuspend in L PBS/BSA (1%). (i) The samples can be directly analyzed under a fluorescence microscope or by flow cytometry. Note: The kit is stable at 15 to 25 C. Note: The enzyme solution (TdT) must be kept on ice and should be discarded after use. Note: The samples can be counterstained with 0.5 g/ml of propidium iodide to provide background DNA staining. Protocol #4: APO-DIRECT kit (BD Pharmingen, Catalog # ) Principal Fragmented DNA can be detected with a reaction catalyzed by exogenous TdT and refereed as end labeling. The assay kit consists of two parts: Part A (Component No. 6536AK) that must be stored at 4 C and part B (Component No. 6536BK) that must be stored at 20 C (Table 14.5). 1. Sample preparation (a) Following liquefaction, load a 5- L aliquot of the sample on a Microcell slide chamber for manual evaluation of concentration and motility. Check the concentration of sperm in the sample. Adjust it to /ml. (b) Suspend the cells in 3.7% (w/v) paraformaldehyde prepared in PBS (ph 7.4). (c) Place the cell suspension on ice for min. (d) Centrifuge to pellet the cells at 300 g for 7 min. Discard the supernatant and suspend the pellet in 1 ml of ice-cold 70% (v/v) ethanol at 20 C until use. Cells can be stored at 20 C several days before use.

10 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 209 Table 14.5 Components of the Apo-direct kit Component No. Size (ml) Description Color code AZ a 25 PI/RNase staining buffer (5 g/ml PI, 200 g/ Amber bottle ml RNase) AZ a 0.50 Reaction buffer (contains cacodylate acid) Green cap (dimethylarsenic) AZ a 100 Rinsing buffer (contains 0.05% sodium azide) Red cap AZ a 100 Wash buffer (contains 0.05% sodium azide) Blue cap EZ b 0.40 FITC-dUTP (0.25 nmol/reaction; contains Orange cap 0.05% sodium azide) LZ b 5 Negative control cells (contains 70% vol./vol. Clear cap ethanol) LZ b 5 Positive control (contains 70% vol./vol. ethanol) Brown cap EZ b TdT enzyme (10,000 U/mg) (20 g/ml in 50% vol./vol. glycerol solution) Yellow cap a Component No. 6536AK to be stored at 4 C b Component No. 6536BK to be stored at 20 C Table 14.6 Preparation of staining solution for the TUNEL test Staining solution 1 assay ( L) 6 assays ( L) 12 assays ( L) Reaction buffer (green cap) TdT enzyme (yellow cap) FITC-dUTP (orange cap) Distilled H 2 O Total volume Staining Protocol (a) Resuspend the positive (6552LZ) and negative (6553LZ) control cells supplied in the kit by swirling the vials. Remove 2-mL aliquots of the control cell suspensions (approximately cells/ml) and place in mm centrifuge tubes. Centrifuge the control cell suspensions for 5 min at 300 g and remove the 70% (v/v) ethanol by aspiration, being careful to not disturb the cell pellet. (b) Resuspend each tube of control and sample tubes with 1.0 ml of Wash Buffer (6548AZ) (Blue cap) for each tube. Centrifuge as before and remove the supernatant by aspiration. (c) Repeat the Wash Buffer treatment. (d) Resuspend each tube of the control cell pellets in 50 L of the Staining Solution (prepared as described below). 3. Staining Solution (single assay) (a) Prepare the staining solution by mixing the appropriate amounts of the staining reagents (Table 14.6). (b) Incubate the sperm in the Staining Solution for 60 min at 37 C. (c) At the end of the incubation time, add 1.0 ml of Rinse Buffer (6550AZ) (Red cap) to each tube and centrifuge each tube at 300 g for 5 min. Remove the supernatant by aspiration. (d) Repeat rinsing with 1.0 ml of Rinse Buffer, centrifuge, and then remove the supernatant by aspiration. (e) Resuspend the cell pellet in 0.5 ml of the PI/RNase Staining Buffer (6551AZ). (f) Incubate the cells in the dark for 30 min at room temperature. (g) Analyze the cells in PI/RNase solution by flow cytometry.

11 210 R. Sharma and A. Agarwal In addition to the negative and positive controls provided with the kit, it is also important to include the negative and positive sperm control samples. Negative control: In this the TdT enzyme is omitted from the reaction mixture. Positive control: DNA damage is induced by adding 100 L of DNase I (1 mg/ml) for 1 h at 37 C. Note: The volume of staining solution needed can be adjusted based on the number of tubes prepared and multiplying with the component volumes needed for one assay. Note: Mix only enough staining solution necessary to complete the number of assays prepared. Note: The staining solution is active for approximately 24 h at 4 C. Note: If the sperm density is low, decrease the amount of PI/RNase Staining Buffer to 0.3 ml. Note: The cells must be analyzed within 3 h of staining. The cells may begin to deteriorate if left overnight before the analysis. Measurement of Sperm DNA Damage Flow Cytometry A minimum of 10,000 events are examined for each measurement at a flow rate of about 100 events/s on a flow cytometer (fluorescence activated cell sorting [FACS]) (Becton and Dickinson, San Jose, CA). The excitation wavelength is 488 nm supplied by an argon laser at 15 mw. Green fluorescence ( nm) is measured in the FL-1 channel and red fluorescence ( nm) in the FL-2 channel. Spermatozoa obtained in the plots are gated using a forward-angle light scatter (FSC) and a side-angle light scatter (SSC) dot plot to gate out debris, aggregates, and other cells different from spermatozoa. TUNEL-positive spermatozoa in the population are measured after converting the data into a histogram (Fig. 14.3). The percentage of positive cells (TUNEL-positive) are calculated on a 1,023-channel scale using the appropriate flow cytometer software (FlowJo Mac version 8.2.4) (FlowJo, LLC, Ashland, OR) as described by us earlier [47] (Fig. 14.3). Fluorescence Microscopy The sperm suspension is counterstained with 4,6 diamidoino-2-phenylindole (DAPI), 2 g/ml in vecta shield (Vector, Burlingame, CA) or propidium iodide (5 L). A minimum of 500 spermatozoa per sample are scored under 40 objective of the epifluorescence microscope. For the green signal (FITC), an excitation wavelength in the range of nm (e.g., 488 nm) and detection in the range of nm are adequate (green). The number of spermatozoa per field stained with DAPI (blue) or PI (red) is first counted and then the number of cells emitting green fluorescence (TUNEL-positive) is counted; and the numbers are expressed as percentage of total count of the sample (Fig. 14.2). Protocol for Shipping Semen Samples for TUNEL Test Semen samples can be shipped to labs that perform the TUNEL assay. Following liquefaction, the sperm count should be checked using these steps: 1. Fixation protocol: (a) Suspend the sperm cells ( cells/ ml) in 3.7% (weight/vol.) paraformaldehyde prepared in PBS (ph 7.4). (b) Place the cell suspension on ice for min. (c) Centrifuge the cells for 5 min at 300 g and discard the supernatant. (d) Adjust the cell concentration to cells/ml in 70% (vol./vol.) ice cold ethanol. (e) Store the cells in 70% (v/v) ethanol at 20 C until use. The cells can be stored at 20 C several days before use. (f) Label the cryovials with the sample information (i.e., date, name, type of sample, volume, etc.). (g) At the time of shipping, place cryovials in the cryoboxes, place these in adequate amount of dry ice and ship it by overnight courier. (h) Enclose the list of the samples being shipped.

12 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 211 (i) Ensure that the quantity of ice is sufficient to last 2 3 days in case of an unexpected delay in delivery. Reference Ranges of Sperm Damage We have established sperm DNA damage reference ranges using the protocol described for the apoptosis detection kit (protocol #4). Unprocessed or raw liquefied seminal ejaculates were used, and healthy donors of proven and unproven fertility were included. A receiver operating characteristic (ROS) curve was used to establish the cutoff values (Fig. 14.4). Normal sperm DNA damage: <20% Abnormal DNA damage: >20% Sensitivity and Specificity The sensitivity of the TUNEL test was 64% with specificity of almost 100%. This cutoff value is specific to our program; other centers should establish their own lab cutoffs, as this will vary with the methodology, assay reagents, staining steps, and patient population (Fig. 14.4). Factors Affecting TUNEL Assay Results The methods used for DNA damage assessment were originally developed and validated for the investigation of DNA in somatic cells. The Fig Receiver operating characteristic (ROC) curve showing cutoff of 19.26%, sensitivity of 64%, and specificity of 100% TUNEL assay includes specific detection of free DNA ends ( nicks ) by enzymatic incorporation of marked nucleotides. Therefore, an important question is whether these adaptations are adequate to allow reagents to access the highly compacted sperm DNA without inducing damage. Owing to a lack of standardization in methods, it is difficult to determine if the variations in the findings are real (related to biology) or due to differences in methods. Therefore, an important question is whether the treatments used to prepare the sperm may themselves induce DNA damage. 1. Accessibility of the DNA. Reagents used in the TUNEL test such as terminal deoxynucleotidyl transferase (TdT) can be a limiting factor. If the protamine bound chromatin is resistant to nucleases, it would be resistant to enzymes such as TdT. However, improperly stabilized protamine and histone bound DNA can be expected to be accessible to TdT as TdT can access DNA breaks in nucleosomal DNA. Therefore, the TUNEL assay can be expected to reveal this type of chromatin structure. 2. Sperm preparation. It is important to understand that the results will be different if the test is performed before or after sperm preparation. This will also influence the predictive potential of assisted reproductive technology (ART) success. The TUNEL assay has been shown to be discriminative for clinical pregnancy using either raw semen or cohorts of spermatozoa prepared by density-gradient centrifugation for clinical use [28, 48]. 3. Presence of dead cells. Interpretation of the results can be confounded by the presence of dead cells as in the case of tests performed on unprocessed semen. Dead cells contain fragmented DNA, and this may bias the overall results. 4. Number of cells examined. A large number of spermatozoa (approximately 400) must be counted for accuracy. If a lower number is counted, the confidence limits will increase. Counting by flow cytometry is faster and more accurate and robust than counting with optical microscopy. In fact, flow

13 212 R. Sharma and A. Agarwal cytometry results were shown to be 2.6 fold higher than the results from fluorescence microscopy [45]. 5. Inter-and intraobserver as well as the inter- and intraassay variations. Establishing inter- and intraobserver as well as inter- and intraassay variations is extremely important [47]. For example, the total variation among a set of healthy normal men (control) would tend to be much less than the total variability among a set of infertile patients, so even if separate observers provided a similar degree of interobserver variability in the two populations with respect to their absolute difference in assigned TUNEL values, the results would look more impressive in the more variable patient sample, since the variance components are compared to total variability. 6. Other factors. Similarly, in establishing the cutoff or normal threshold values as well as the sensitivity, specificity, NPV and PPV, it is important to determine whether a test will be utilized as a screening/diagnostic test or to predict an established end point. Sensitivity is important and must be high for a test to be used for screening or diagnostic purposes so that it can be offered to a large population. However, specificity becomes critical for a test to be offered as a predictive marker of a defined end point. Positive and negative predictive values are dependent on the prevalence of infertility in the tested population, so they will be different in populations where the percentage of fertile subjects may be higher [47]. Sergerie et al. [38] examined the threshold values of TUNEL in 47 men with proven fertility and in 66 infertile men. The infertile men had higher mean level of DNA damage than the proven fertile men (40.9 ± 14.3% vs ± 7.3%) (P < 0.001). The area under curve was 0.93 for a 20% cutoff; the specificity was 89.4% and the sensitivity was 96.9%. The positive and negative predictive values were 92.8 and 95.5%, respectively. Our study [47] with 194 infertile patients and a control population consisting of men with proven and unproven fertility showed a very similar cutoff value (19.2 vs. 20%) to those reported by Sergerie et al. [38]. These values are similar to those reported earlier (20%; [31] and 24.3%; [49]; and 24%; [38]). These values are much lower than the threshold established for SCSA ( 30%). Both sensitivity and specificity are associated with intrinsic performance of the TUNEL assay. However, the PPV and NPV are strongly associated with the prevalence of the sample [38]. Standardized methods that allow researchers to compare results from different laboratories are needed. It is important to understand and control for changes in sperm chromatin that occur after ejaculation and to distinguish between genuine and artifactual variations caused by a lack of reagent access to DNA. Standardized protocols and appropriate external quality controls are necessary to implement findings worldwide. For useful clinical cutoff limits, it is also necessary that the test can distinguish between affected and unaffected individuals. In this context, correlations by themselves are not adequate and other means of interpreting results such as predictive value, likelihood ratios, odds ratios, ROS cutoff value are more valuable [50]. Future of TUNEL Assay The literature suggests that this test is a safe and effective means of measuring sperm DNA damage. Additional research needs to be generated to further fine tune the lower thresholds and minimize the variations in the methodology. The highly specialized and compacted nature of sperm chromatin makes it less permeable and less sensitive to allow the terminal enzyme (TdT) to access the DNA stand breaks deep within the sperm nucleus. This particular issue may be one factor that contributes to variation in results. To overcome this challenge, a recent study [51] has used Dithiothreitol (DTT) to expose the chromatin for 45 min prior to the fixation step. This simple additional step significantly improved the signals generated by the spermatozoa. Furthermore, the TUNEL methodology was refined to include a vitality stain (Live/Dead Fixable Dead cell stain)

14 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 213 that remained associated with the spermatozoa during fixation and processing of the TUNEL assay, thereby allowing both DNA integrity and vitality to be simultaneously detected in the same flow-cytometry assay. This modification allows the assay to be more sensitive and robust. Measuring viability in the spermatozoa tested for DNA damage by TUNEL is critical, as this may help further improve the predictive value of this test. Conclusions Sperm DNA integrity is essential for the accurate transmission of genetic information. Sperm chromatin is a highly specialized and compact structure that is essential for protection and transmission of the human genome. A large number of tests are available to assess different aspects of sperm DNA integrity, but there is no consensus on the optimal technique or appropriate clinical cutoff levels. We review the use of TUNEL test by flow cytometry and fluorescence microscopy as used by different laboratories, its advantages and challenges and highlight further improvements to make it more robust. This test has the potential of being offered to a select group of infertile patients presenting with idiopathic infertility or in cases where oxidative stress may be an underlying issue. The use of this test can be cost-effective in establishing the DNA integrity of the sperm in selected cases of male infertility by any fertility testing facility with access to flow cytometry before considering other more expensive ART procedures. Further research is needed to create a platform for andrology labs and other testing centers to use with this test in measuring sperm DNA damage. References 1. Aitken RJ, De Iuliis GN. On the possible origins of DNA damage in human spermatozoa. Mol Hum Reprod. 2010;16: Ollero M, Gil-Guzman E, Lopez MC, et al. Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility. Hum Reprod. 2001;16: Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, Nelson DR, Thomas AJ, Jr. Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril. 2003;79: Aitken RJ, Sawyer D. The human spermatozoon-not waving but drowning. Adv Exp Med Biol. 2003; 518: De Iuliis GN, Thomson LK, Mitchell LA, Finnie JM, Koppers AJ, Hedges A, Nixon B, Aitken RJ. DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2 -deoxyguanosine, a marker of oxidative stress. Biol Reprod. 2009;81: Sakkas D, Mariethoz E, Manicardi G, et al. U. Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod. 1999;4: Marcon L, Boissonneault G. Transient DNA strand breaks during mouse and human spermiogenesis new insights in stage specificity and link to chromatin remodeling. Biol Reprod. 2004;70: Sakkas D, Seli E, Bizzaro D Tarozzi N, Manicardi GC.. Abnormal spermatozoa in the ejaculate: abortive apoptosis and faulty nuclear remodelling during spermatogenesis. Reprod Biomed Online 2003;7: Weng SL, Taylor SL, Morshedi M, Schuffner A, Duran EH, Beebe S, Oehninger S. Caspase activity and apoptotic markers in ejaculated human sperm. Mol Hum Reprod. 2002;8: Matsuda Y, Tobari I, Maemori M, Seki N. Mechanism of chromosome aberration induction in the mouse egg fertilized with sperm recovered from postmeiotic germ cells treated with methyl methanesulfonate. Mutat Res. 1989;214: Aitken RJ. Founders Lecture. Human spermatozoa: fruits of creation, seeds of doubt. Reprod Fertil Dev. 2004;16: Steele EK, Lewis SE, McClure N. Science versus clinical adventurism in treatment of azoospermia. Lancet. 1999;13;353: O Connell M, McClure N, Powell LA, Steele EK, Lewis SE. Differences in mitochondrial and nuclear DNA status of high-density and low-density sperm fractions after density centrifugation preparation. Fertil Steril. 2003;79: Lewis SE, O Connell M, Stevenson M, Thompson- Cree L, McClure N. An algorithm to predict pregnancy in assisted reproduction. Hum Reprod. 2004;19: Suganuma R, Yanagimachi R, Meistrich ML. Decline in fertility of mouse sperm with abnormal chromatin during epididymal passage as revealed by ICSI. Hum Reprod. 2005;20: Sailer BL, Jost LK, Evenson DP. Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured

15 214 R. Sharma and A. Agarwal by the terminal deoxynucleotidyl transferase assay. J Androl. 1995;16: Zini A, Sigman M. Are tests of sperm DNA damage clinically useful? Pros and cons. J Androl. 2009;30: Muratori M, Piomboni P, Baldi E, Filimberti E, Pecchioli P, Moretti E, Gambera L, Baccetti B, Biagiotti R, Forti G, Maggi M. Functional and ultrastructural features of DNA-fragmented human sperm. J Androl. 2000;21: Muratori M, Maggi M, Spinelli S, Filimberti E, Forti G, Baldi E. Spontaneous DNA fragmentation in swim-up selected human spermatozoa during long term incubation. J Androl. 2003;24: Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril. 1998;69: Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod. 2000;15: Donnelly ET, O Connell M, McClure N, Lewis SE. Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa. Hum Reprod. 2000;15: Gandini L, Lombardo F, Paoli D, Caponecchia L, Familiari G, Verlengia C, Dondero F, Lenzi A. Study of apoptotic DNA fragmentation in human spermatozoa. Hum Reprod. 2000;15: Oosterhuis GJ, Mulder AB, Kalsbeek-Batenburg E, Lambalk CB, Schoemaker J, Vermes I. Measuring apoptosis in human spermatozoa: a biological assay for semen quality? Fertil Steril. 2000;74: Sergerie M, Ouhilal S, Bissonnette F, Brodeur J, Bleau G. Lack of association between smoking and DNA fragmentation in the spermatozoa of normal men. Hum Reprod. 2000;15: Ramos L, Wetzels AM. Low rates of DNA fragmentation in selected motile human spermatozoa assessed by the TUNEL assay. Hum Reprod. 2001;16: Zini A, Bielecki R, Phang D, Zenzes MT. Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertil Steril. 2001a;75: Duran EH, Morshedi M, Taylor S, et al. Sperm DNA quality predicts intrauterine insemination outcome: a prospective cohort study. Hum Reprod. 2002;17: Sakkas D, Moffatt O, Manicardi GC, Mariethoz E, Tarozzi N, Bizzaro D. Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biol Reprod. 2002;66: Shen HM, Dai J, Chia SE, Lim A, Ong CN. Detection of apoptotic alterations in sperm in subfertile patients and their correlations with sperm quality. Hum Reprod. 2002;17: Benchaib M, Braun V, Lornage J, et al. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod. 2003;18: Carrell DT, Liu L, Peterson CM, Jones KP, Hatasaka HH, Erickson L, Campbell B. Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss. Arch Androl. 2003;49: Erenpreisa J, Erenpreiss J, Freivalds T, Slaidina M, Krampe R, Butikova J, Ivanov A, Pjanova D. Toluidine blue test for sperm DNA integrity and elaboration of image cytometry algorithm. Cytometry. 2003;52: Erenpreiss J, Jepson K, Giwercman A, Tsarev I, Erenpreisa J, Spano M. Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays. Hum Reprod. 2004; 19: Lachaud C, Tesarik J, Cañadas ML, Mendoza C. Apoptosis and necrosis in human ejaculated spermatozoa. Hum Reprod. 2004;19: Tesarik J, Greco E, Mendoza C. Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation. Hum Reprod. 2004;19: Greco E, Iacobelli M, Rienzi L, Ubaldi F, Ferrero S, Tesarik J. Reduction of the incidence of sperm DNA fragmentation by oral antioxidant treatment. J Androl. 2005;26: Sergerie M, Laforest G, Bujan L, et al. Sperm DNA fragmentation: threshold value in male fertility. Hum Reprod. 2005;20: Stahl O, Eberhard J, Jepson K, Spano M, Cwikiel M, Cavallin Stahl E, Giwercman A. The impact of testicular carcinoma and its treatment on sperm DNA integrity. Cancer 2004;100: Sépaniak S, Forges T, Monnier-Barbarino P. Cigarette smoking and fertility in women and men. Gynecol Obstet Fertil. 2006;34: Chohan KR, Griffin JT, Lafromboise M, De Jonge CJ, Carrell DT. Comparison of chromatin assays for DNA fragmentation evaluation in human sperm. J Androl. 2006;27: de Paula TS, Bertolla RP, Spaine DM, Cunha MA, Schor N, Cedenho AP. Effect of cryopreservation on sperm apoptotic deoxyribonucleic acid fragmentation in patients with oligozoospermia. Fertil Steril. 2006; 86: Aoki VW, Emery BR, Liu L, Carrell DT. Protamine levels vary between individual sperm cells of infertile human males and correlate with viability and DNA integrity. J Androl. 2006;27: Spermon JR, Ramos L, Wetzels AM, Sweep CG, Braat DD, Kiemeney LA, Witjes JA. Sperm integrity pre- and post-chemotherapy in men with testicular germ cell cancer. Hum Reprod. 2006;21: Domínguez-Fandos D, Camejo MI, Ballescà JL, Oliva R. Human sperm DNA fragmentation: correlation of TUNEL results as assessed by flow cytometry and optical microscopy. Cytometry. 2007;71: Sakamoto Y, Ishikawa T, Kondo Y, Yamaguchi K, Fujisawa M. The assessment of oxidative stress in

16 14 Laboratory Evaluation of Sperm Chromatin: TUNEL Assay 215 infertile patients with varicocele. BJU Int. 2008;101: Sharma RK, Sabanegh E, Mahfouz R, Gupta S, Thiyagarajan A, Agarwal A. TUNEL as a test for sperm DNA damage in the evaluation of male infertility. Urology 2010;76: Borini A, Tarozzi N, Bizzaro D, Bonu MA, Fava L, Flamigni C, Coticchio G. Sperm DNA fragmentation: paternal effect on early post-implantation embryo development in ART. Hum Reprod. 2006;21: Henkel R, Kierspel E, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, Schill WB, Kruger TF. DNA fragmentation of spermatozoa and assisted reproduction technology. Reprod Biomed Online. 2003;7: Barratt CL, Aitken RJ, Björndahl L, Carrell DT, de Boer P, Kvist U, Lewis SE, Perreault SD, Perry MJ, Ramos L, Robaire B, Ward S, Zini A. Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications a position report. Hum Reprod. 2010;25: Mitchell LA, De Iuliis GN, Aitken RJ. The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influenced by DNA compaction and cell vitality: development of an improved methodology. Int J Androl. 2011;34:2 13.

17 Basic and Clinical Aspects 15 of Sperm Comet Assay Luke Simon and Sheena E.M. Lewis Abstract Sperm DNA damage is associated with poorer assisted reproductive treatment (ART) outcomes including reduced fertilization rates, embryo quality, and pregnancy rates and higher rates of spontaneous miscarriage and childhood diseases. It shows promise as a more robust biomarker of infertility than conventional semen parameters. Among the sperm DNA testing methods, the alkaline comet assay is a sensitive, reliable, and powerful tool to detect even low levels of DNA damage within individual sperm. The present chapter provides an overview of the use of the alkaline comet assay in sperm. This includes the need for standardization of the alkaline comet assay protocol and its present strengths and weaknesses. Since sperm DNA damage is often the result of increased oxidative stress in the male reproductive tract, primarily formed due to an imbalance between reactive oxygen species generation and antioxidant depletion, a novel addition to the comet assay to measure oxidized bases is explored. The potential use of antioxidant therapy to protect against such damage is also described. Finally, the diagnostic and prognostic values of sperm DNA damage measures in determining the assisted reproductive technology (ART) success are discussed. Keywords S.E.M. Lewis ( ) Centre for Public Health, Queen s University of Belfast, Room 208, Institute of Clinical Science, Belfast, Northern Ireland, UK s.e.lewis@qub.ac.uk The Need for Novel Diagnostic and Prognostic Tests Male infertility is implicated in more than 40% of couples presenting for treatment with assisted reproductive technology (ART). Conventional A. Zini and A. Agarwal (eds.), Sperm Chromatin: Biological and Clinical Applications in Male Infertility and Assisted Reproduction, DOI / _15, Springer Science+Business Media, LLC

18 218 L. Simon and S.E.M. Lewis semen analysis continues to be the only routine test to diagnose male infertility. However, semen analysis cannot discriminate between the sperm of fertile and infertile men [1]. Recent evidence has suggested that instability in the genomic material of the sperm nuclei is a more robust parameter in measuring the fertility potential of sperm, either in vivo or in vitro. For a test to be useful diagnostically or prognostically, it must have a threshold value that provides a discriminatory power above or below the threshold value with little overlap between groups of fertile and infertile men and couples with ART success and failure. However, neither the routine semen analysis nor the available sperm DNA tests yet meet these standards (reviewed in references [2, 3]). The primary function of the sperm is to deliver the paternal genome to the oocyte. Recent studies have shown a number of sperm nuclear abnormalities such as DNA strand breaks, Y chromosome microdeletions, alterations in chromosome number, distorted epigenetic regulation and sperm s environmental milieu during epididymal transport and ejaculation. Factors such as increased oxidative stress or low levels of antioxidants may have implications on male reproductive health [4]. As the structural organization of the sperm chromatin is also essential for the normal function of the sperm [5], character ization of sperm DNA quality has gained importance. In recent years, comet assay, TUNEL, SCSA, and SCDA or Halo assay, in situ nick end labeling have been studied extensively to analyze sperm chromatin integrity. Each of these tests determines different aspects of DNA integrity, but to date, combining all the studies available in metaanalysis shows that these tests lack the statistical power and diagnostic potential necessary to incorporate them into routine clinical use. Causes of Sperm DNA Damage In recent years, the generation of reactive oxygen species (ROS) has been widely studied in the male reproductive tract and reported to be a concern because of their toxic effects on sperm quality and function (reviewed by Saleh and Agarwal [6]). They have been shown to cause DNA fragmentation in the reproductive tract as well as damage in ejaculated sperm [7]. High levels of ROS have also been reported in the seminal plasma of infertile men [8]. Sperm are vulnerable to the oxidative-stress-mediated damage, due to their structure with a high proportion of polyunsaturated fatty acids in their plasma membranes [9]. As sperm cannot repair such damage, sperm DNA has evolved to protect itself by compact packaging of the sperm DNA by protamines [10, 11]. The exact mechanisms by which ROS induces DNA damage are poorly understood, However, ROS-induced sperm DNA damage is exemplified by DNA cross-links, frameshifts, production of base free sites, chromosomal rearrangements and DNA base-pair oxidation [12 14]. It is also well known to cause strand breaks, with the levels of ROS correlated with increased percentage of single and double-strand damage in sperm [15 17]. ROS-mediated DNA damage is also seen in the formation of modified bases, which are often converted into strand breaks and considered to be important biomarkers for oxidative DNA damage [18]. Finally, ROS cause gene mutations such as point mutations and polymorphism [19, 20]. Seminal plasma is contaminated with ROS [21, 22] primarily produced by leukocytes and defective sperm [23]. The presence of elevated levels (> /ml) of leukocytes in the semen is defined as leukocytospermia [24] and is associated with increased levels of ROS, leading to sperm DNA damage [25]. Cytoplasmic droplets are also associated increased ROS generation and poor sperm quality [26, 27]. Environmental and Lifestyle Hazards It has recently been reported that male fertility declines with age, even though spermatogenesis continues [28]. An increase in male age has been associated with increased genetic and chromosomal defects [29, 30]. Men over 37 years have been shown to three times more sperm DNA